Biologically active drug polymer derivatives and method for preparing same

ABSTRACT

New biologically active drug polymer derivatives, namely peptides or protein derivatives, are useful medicaments and are represented by the generic formula: 
     
         RO--(CH.sub.2 --CH.sub.2 O).sub.n --(CO)--NH--X--(CO)--NH--Z (I) 
    
     wherein 
     R represents a lower alkyl group, 
     n is an integer comprised between 25 and 250, 
     X when combined with adjacent NH and CO groups represents an amino acid or a dipeptide or tripeptide residue, and 
     Z when combined with the adjacent NH group represents a biologically active peptide or protein or NH or NH 2  containing drug residue.

This is a continuation of application Ser. No. 07/681,493, filed Jun. 3,1991, now abandoned.

The invention relates to new biologically active drug polymerderivatives, namely, peptides or protein derivatives useful asmedicaments. It relates more particularly to peptide or proteinpolyethylene glycol derivatives wherein the peptide or protein moeity islinked to the polyethylene glycol residue by means of an amino acid orpeptide spacer arm.

Modification of biologically active substances such as peptides orproteins with monomethoxy polyethylene glycol is reported to changeextensively their physical, chemical, enzymological, immunological, aswell as their pharmacological and pharmacokinetic properties. Severalmethods to achieve such a modification have so far been reported (seee.g. U.S. Pat. Nos. 4,179,337 and 4,766,106; Appl. Biochem andBiotechnology, Vol. 11, p. 141/1985).

Such modified peptide or protein derivatives exhibit some advantageswhen compared to the peptide or protein itself: increased watersolubility, decreased antigenicity or increased half-life of thecirculating peptide or protein.

The use of such modified bioactive compounds, however, is not satisfyingas the following drawbacks have been observed: difficulty to obtain aselective incorporation of a radioactive probe into the polymer drugadduct necessary for pharmacokinetic experiments; inactivation of someenzymes; difficulty to program (or to modulate) the cleavage of thepolymer-protein bond by specific enzymes in the body; difficulty ofintroduction into the polymerdrugs adduct amino acid sequences which mayconfer targeting properties to the adducts itself. These disadvantagesare related to the chemistry employed in the polymer activation and toits direct linkage to the drug.

It has been found that some, if not all of the above mentioned drawbackscan be eliminated or at least significantly reduced by making use of thenew drug polymer derivatives of the invention which are represented bythe generic formula

    RO--(CH.sub.2 --CH.sub.2 O)n--(CO)--NH--X--(CO)--NH--Z     (I)

wherein

R represents a lower alkyl group,

n is an integer comprised between 25 and 250,

X when combined with adjacent NH and CO groups represents an amino acidor a dipeptide or tripeptide residue, and

Z when combined with the adjacent NH group represents a biologicallyactive peptide or protein or NH or NH₂ -containing drug residue.

Preferred species of compounds of formula (I) are those wherein Rrepresents a methyl group and wherein n is an integer comprised between40 and 115, i.e. those of which the polyethylene moiety exhibits amolecular weight of about 1800 to 5500, for example of 1900 and 5000.

Also preferred are the compounds of formula (I) wherein symbol X whencombined with the adjacent NH and CO groups represents an amino acidselected from glycine, phenylylanine, tryptophan and norleucine, or adipeptide or tripeptide such as Gly-Gly, Arg-Arg, Phe-Arg, Gly-Gly-Arg,Gly-Gly-Phe or Gly-Leu-Gly-Leu.

Also preferred are the compounds of formula (I) wherein symbol Z, whencombined with the adjacent NH group represents the residue of abiologically active peptide, protein or drug, selected from thefollowing species:

enzymes such as superoxidedismutase, ribonuclease, arginase,asparaginase, urokinase, e.g.;

antibiotics such as ampicillin, doxorubicin e.g.;

synthetic drugs like N-desmethyl-tamoxifen;

peptides such as LHRH and synthetic analogues of same, somatostatin andsynthetic analogues of same, e.g.;

proteins such as interleukin-2, tumor necrosis factor, insulin, IGF-1e.g.;

nucleosides such as adenin-arabinoside (ara-A), cytosin-arabinoside(ara-C), acyclovir e.g.

This enumeration is in no way limitative.

Some but not all of the most interesting peptide derivatives of formula(I) are mentioned and characterized individually in the Examples.

Consequently, the invention relates to new biologically active peptidederivatives of formula (I) as defined above, as well as to a method forpreparing same.

The invention also relates to pharmaceutical compositions which compriseat least one of the compounds of formula (I) as active ingredient.Further objects of the invention shall appear from the specification orthe claims.

The method of the invention is based on the linkage of an amino acid orpeptide spacer arm of various structures and properties to the hydroxylfunction of monoalkoxypolyethylene glycol through a carbonate linkagewhich involves the NH₂ group of the amino acid or peptide. This reactionis followed by the activation of the COOH function of the amino acid orpeptide spacer arm as succinimidyl ester which, thus, becomes reactivetowards the amino group of the biologically active peptide, protein ordrug.

More specifically the method of the invention consists of:

a) reacting a mono-alkoxy-polyethylene glycol derivative of formula

    RO--(CH.sub.2 --CH.sub.2 O).sub.n H                        (II)

wherein R and n have the definition provided above, with2,4,5-trichlorphenylchloroformate or 4-nitrophenylchloroformate toobtain the corresponding carbonate;

b) reacting the carbonate thus obtained with an amino acid or a di- ortripeptide of formula

    H.sub.2 N--X--(CO)OH                                       (III)

wherein X is defined above to obtain a compound of formula

    RO--(CH.sub.2 --CH.sub.2 O).sub.n --(CO)--NH--X--(CO)OH    (IV)

c) converting the compound of formula (IV) thus obtained into thecorresponding succinimidyl ester, and

d) finally, reacting the said succinimidyl ester with a biologicallyactive peptide or protein or NH or NH₂ -containing drug of formula

    R--NH--Z or H.sub.2 N--Z                                   (V)

wherein R and Z are defined as indicated above.

Steps a) through d) of the above described method do not necessitatespecial reaction conditions and can be carried out according to theusual techniques. Details of each of the above reaction steps areprovided in the Examples illustrating the invention.

By means of the introduction of such a new spacer arm (amino acid orpeptide) an improved targeting of the bioactive protein or drug isachieved: an enhanced lyposomal degradation of the peptide derivative offormula (I), a site-specific cleavage of the derivative by specificcellular enzymes as well as, in some instances, an increased binding ofthe derivative to specific cellular receptors which recognize the aminoacid moiety.

There are still additional advantages: the new spacer arm may contain aresidue which can be conveniently used to quantitate directly thepolymer chains introduced into the protein. This can be performed by UVabsorption in the case of tryptophan or phenylalanine, or by amino acidanalysis in the case of norleucine which is not naturally present inproteins from natural sources.

The spacer arm may also be made radioactive using labelled amino acids,which simplifies to a great extent the detection of the biologicallyactive peptide derivative during pharmacokinetic or metabolicexperiments.

Some of these interesting properties are illustrated in the followingExamples which are not limitative. In the said Examples the term "M-PEG"defines a monomethoxypolyethylene glycol and the amino acids or peptidesare described by means of the terms usual in the art.

FIG. 1 illustrates a chromatograph of the amino acid tryptophan modifiedwith monomethoxypolyethylene glycol (M-PEG).

FIG. 1a illustrates a chromatograph of the amino acid tryptophan.

FIG. 2 illustrates a chromatograph of the amino acid phenalaninemodified with monomethoxypolyethylene glycol (M-PEG).

FIG. 2a illustrates a chromatograph of the amino acid phenalanine.

FIG. 3 illustrates a chromatograph of the amino acid tryptophan modifiedwith M-PEG and superoxide dismutase (SOD).

FIG. 3a illustrates a chromatograph of the amino acid glycine modifiedwith M-PEG and superoxide dismutase (SOD).

A. Preparation of activated M-PEG with an amino acid or peptide spacerarm EXAMPLE 1 M-PEG 5000-Gly-Succinimidyl ester (M-PEG 5000-Gly-OSu)

To 10 g (2 mM) of M-PEG-5000, dissolved in 50 ml of anhydrous methylenechloride, 0.56 ml (4 mM) of triethylamine (TEA) and 0.81 g (4 mM) of4-nitrophenyl chloroformate were added under stirring while the pH wasadjusted at 7.5-8.0 with TEA. The reaction mixture was maintained atroom temperature for 4 hrs. The mixture, concentrated under vacuum toabout 10 ml, was dropped into 200 ml of stirred diethyl ether. Theprecipitate was collected by filtration and crystallized twice from hotethyl acetate. The yield of M-PEG-p-nitrophe-nylcarbonate(M-PEG-OCO-OPh-NO₂), calculated spectrophotometrically on the basis ofp-nitrophenol absorption was over 95%.

Glycine 1.5 g (20 mM) were dissolved in 20 ml of water, the solution wasadjusted to pH 8.0-8.3 and added under stirring of 10.33 g (2 mM) ofM-PEG-OCO-O-Ph-NO₂ while the pH was maintained at 8.3 with NaOH. After 4hrs at room temperature the solution, cooled at 0° C. and brought to pH3 with 2N HCl, was extracted three times with CHCl₃. The chloroform waswashed with water, dried with Na₂ SO₄, concentrated, precipitated withdiethyl ether and the precipitate recrystalized from ethanol. The yield,calculated by COOH titration and glycine evaluation by conventionalamino acid analysis after acid hydrolysis, was 85%.

M-PEG-Gly-OH 10.2 g (2 mM) was dissolved in 50 ml of anhydrous methylenechloride, cooled to 0° C., and 0.46 g (4 mM) of N-hydroxysuccinimide and0.83 g (4 mM) of N,N-dicyclohexylcarbodiimide were added under stirring.The stirring was continued for 4 hrs, while the temperature was raisedto 20° C. The precipitated dicyclohexylurea was removed from thereaction mixture by filtration, the solution was concentrated undervacuum and the product precipitated with diethyl ether andrecrystallized from ethyl acetate. The yield of esterification,calculated from the UV hydroxysuccinimide absorption, was 85%.

Starting from M-PEG 1900 the M-PEG-1900-Gly-OSu derivative was obtainedfollowing the same procedure with a similar yield.

EXAMPLE 2 M-PEG 5000-Trp-succinimidyl ester (M-PEG 5000-Trp-OSu)

The procedure described above gave the PEG-tryptophan derivative with ayield of 80% calculated on the basis of the hydroxysuccinimideabsorption as well as the tryptophan absorption at 280 nm (FIG. 1a).

The product presented the characteristic tryptophan absorption spectraas reported in FIG. 1.

EXAMPLE 3 M-PEG 5000-Phe-succinimidyl ester (M-PEG 5000-Phe-OSu)

Following the procedure reported in Example 1 the M-PEG phenylalaninederivative was obtained. The product gave the spectra reported in FIG. 2with the typical phenylalanine absorption at 260 nm (FIG. 2a).

EXAMPLE 4 M-PEG-nor-Leu-succinimidyl ester (M-PEG-5000-nor-Leu-OSu)

This derivative was obtained as above described with both M-PEG 5000 andM-PEG 1900. The 95% yield was calculated by nor-Leu evaluation on anamino acid analyzer after acid hydrolysis.

EXAMPLE 5 M-PEG 5000-Gly-Gly-succinimidyl ester (M-PEG-5000-Gly-Gly-OSu)

Using Gly-Gly as a model compound, the procedure already described underExample 1 was followed to prepare an activated monomethoxy polyethyleneglycol with a dipeptide as a spacer arm. The product, crystallized fromethyl acetate, was obtained with a 85% yield.

B. Bioactive substances modification with amino acid derivatized M-PEGEXAMPLE 6 Superoxide dismutase modification

6.1. With M-PEG 5000-Gly-OSu

Yeast superoxide dismutase (SOD, EC 1.15.1.1.) (100 mg) were dissolvedin 10 ml of borate buffer 0.2 M pH 8 and 640 mg of M-PEG 5000-Gly-OSuwere added at room temperature under vigorous stirring while the pH wasmaintained. The mixture was left standing for 30 min.

The extent of linked polymer chains, determined on the basis of aminogroups modification evaluated according to the method oftrinitrophenylation of Snyder and Sabocinski (Snyder S. I. andSabocinsky P. Z., Anal. Biochem, 64 248-288, 1975) was over 85-90% whilea 20% reduction in enzymatic activity was observed. The enzyme wasevaluated by the method of Paoletti et al. (Paoletti F., Aldinicci D.Mocali A. and Caparrini A., Anal. Biochem., 154 536-541, 1986).

The excess of polymer was removed by twice ultrafiltration on a PM 10AMICON membrane and the concentrated enzyme chromatographied on aBIO-GEL A 0.5 m column. The M-PEG modified enzyme is eluted first assymetrical peak as revealed by UV absorption (FIG. 3a), iodine reactionfor M-PEG and enzymatic activity. The excess of M-PEG is eluted laterfollowed by the leaving group hydroxysuccinimide. The protein peakfractions are collected and lyophylized after membrane ultrafiltration.The M-PEG modified SOD is stored at 0° C. in a dessicator.

6.2- With M-PEG-5000-Trp-OSu

The reaction was carried out as reported above (see 6.1); a similarextent of linked polymer chains to SOD and enzyme activity reduction wasobserved while the product presented the spectrum reported in FIG. 3where the contribution of tryptophan is evident.

6.3- With M-PEG 5000-nor-Leu-OSu

The reaction carried out as reported in 6.1 gave a product with similarenzymatic properties and extent of modification by TNBS assay. In thiscase the amino acid analysis after acid hydrolysis revealed the presenceof norleucine which accounted for 18 M-PEG chains bound to each SODmolecule in agreement with the TNBS test.

6.4- With M-PEG 1900-Gly-OSu

The reaction was carried out as in 6.1, similar results were obtained asfar as polymer linkage and enzymatic activity is concerned, this productis eluted later from the column as expected from the lower molecularweight of the polymer used in the modification.

Comment to examples 6.1 through 6.4: the purification from unreactedM-PEG 5000 or M-PEG 1900 could be successfully reached by dilution ofthe reaction mixture (about 1 to 10 folds) followed by ultrafiltrationconcentration on an AMICON membrane; this procedure of dilution andultrafiltration must be repeated at least 4 times.

Pharmacokinetic behavior of native and M-PEG-modified SOD

Unmodified yeast superoxide dismutase (5.5 mg) or equiactive amount ofSOD modified with M-PEG 5000-Gly or M-PEG 1900-Gly were injected intothe tail vein of Wistar albine male rats.

On a scheduled time the blood was removed by heart puncture withheparinized siringe and SOD evaluated in the plasma on the basis of itsenzymatic activity. Before activity evaluation in plasma was purifiedfrom interferences by CM cellulose and SEPHADEX G 25 columnchromatography. A 50 % clearance of 6 min, 15 and 28 hrs wasrespectively found for the native, the M-PEG 1900 and M-PEG 50000modified derivatives.

Enzymatic properties

The stability of the M-PEG 1900 and M-PEG 5000 modified yeast superoxidedismutase to different conditions are as follows:

a. The M-PEG modified enzyme is less stable to incubation in a proteindenaturant such 2M guanidinium chloride; after 4 hrs its residualactivity is 10% in comparison to the 20% of the native enzyme.

b. The M-PEG 5000-Gly-SOD was maintained in water at a concentration of1 mgml at 0°, 20° or 35° C. No loss of activity was found for at least 8days incubation. The stability was also observed after 8 days standingat 20° C. at a concentration as low as 0.01 mg/ml.

The M-PEG 5000-Gly-SOD was found to be stable to repeated freezing andthawing cycles.

A M-PEG enzyme solution was evaporated to dryness at low temperatureunder vacuum, dissolved and again concentrated; the M-PEG modifiedenzyme was stable for at least six of such cycles while the unmodifiedenzyme lost at least 15% of its activity under the same conditions.

The M-PEG 5000-Gly-SOD was completely stable to repeated cycles ofdissolution and lyophilization whereas the free enzyme at each treatmentlost about 5% of its activity.

The M-PEG 5000-Gly-SOD, in the presence of metal chelates, was found tolose with greater difficulty the metals essential for the activity ascompared to the free enzyme.

EXAMPLE 7 Arginase modification (M-PEG 5000-Gly-arginase

Bovine liver arginase (EC 3.5.3.1), 100 mg, highly purified according toliterature to give a specific activity of 1900 IU/mg, was dissolved in15 ml of carbonate buffer pH 8.5, 0.2M and 800 mg of M-PEG 5000-Gly-OSuwere added under vigorous stirring while the pH was maintained by apH-stat with NaOH 0.1N in a microburette. After 30 minutes the solutionwas diluted to 50 ml with water and ultrafiltered at 4° C. with anAMICON PM 10 ultrafiltration membrane to reduce the volume to about 5ml. The M-PEG modified arginase was purified from excess reagent andby-products of reaction through column chromatography as reported inExample 1. The binding of polymer was at the level of over 50% ofarginase amino groups while only a 5% reduction in arginase activity wasdetected.

Enzymatic and pharmacokinetic properties of M-PEG-5000-Gly-arginase

The modification increased the stability of the enzyme to the action ofproteolyctic enzymes such trypsin, chimotrypsin, elastase andsubtilysin.

The pharmacokinetic behavior of native and PEG derivatized enzyme wasevaluated in the rats as reported under example 6.1. A 50% clearancetime of 1.5 and 8 hrs was respectively found for the unmodified and thepolymer modified arginase.

EXAMPLE 8 Ribonuclease modification (M-PEG 5000-Gly-ribonuclease)

Ribonuclease A (EC 2.7.7.16) from bovine pancreas was modified andpurified as in example 6.1. The amount of M-PEG-Gly-OSu used for themodification was at a molar ratio of 2.5:1 calculated on the availableamino groups of the enzymes. The modification resulted in the covalentlinkage of 11 molecules of polymer for ribonuclease molecule.

The modification is accompanied by an enzyme activity loss of about 10%as verified with cytidine-2',3'-cycle phosphate while the modifiedenzyme was found to be 50% active towards ribonucleic acid.

EXAMPLE 9 Urokinase modification (M-PEG-500-Gly-urokinase)

Urokinase (EC 3.4.4.a) from urine was modified and purified as reportedunder example 6.1. With this enzyme the modification was carried outusing a molar ratio of activated polymer/protein amino group of 1:2.Under these circumstances about 10 molecules of polymer were linked toeach urokinase molecule. The enzymatic activity evaluated on the lysisof thrombus was 30% of that of the native enzyme while its esteroliticactivity, assayed on the synthetic substratecarbobenzoxy-lysine-O-nitrophenyl ester, was the same of the unmodifiedurokinase.

EXAMPLE 10 Ampicillin modification 10.1 - M-PEG 5000-Gly-Ampicillin

To a solution of ampicillin sodium salt, 50 mg (0,135 mM) in 5 ml ofborate buffer 0,2M pH 8, 600 mg (0,12 mM) of M-PEG 5000-Gly-OSu wereadded under vigorous stirring.

The reaction mixture was left standing for 20 min, then separated byexcess of ampicillin and of side products of reaction by gel filtrationchromatography on a BIO GEL P 60 100-200 mesh column. The M-PEG modifieddrug was eluted first as a symmetric peak as revealed the UV absorptionof ampicillin and the iodine reaction for PEG.

The drug modified peak fractions were collected, concentrated byultrafiltration and lyophilized. The product was crystallized from ethylacetate with a 70% yield based on the starting ampicillin. The sameproduct was also prepared by the procedure that is reported below.

10.2 - M-PEG 5000-Gly-Ampicillin

Ampicillin sodium salt 100 mg (0.27 mM) were solved in 20 ml ofN,N-dimethylformamide (DMF); 1.0 g (0.2 mM) of M-PEG 5000-Gly-OSu and0.03 ml of 4-methylmorpholine (NMM) were added while pH was adjusted at8-8.3 with NMM. The reaction mixture was maintained at room temperatureunder stirring for about 4 hrs and then concentrated to dryness underhigh vacuum. The residue was solved in 5 ml of CH₂ Cl₂ which weredropped in stirred diethyl ether (100 ml). The precipitate was removedby filtration and crystallized. The first crystallization was from hotethyl acetate and the second one from hot methanol. The yield, based onthe starting ampicillin, was 60%.

EXAMPLE 11 Doxorubicin modification (M-PEG 5000-Gly-doxorubicin)

To a solution of doxorubicin hydrocloride, 50 mg (8.6.10⁻² mM) of M-PEG5000-Gly-OSu were added in portions. The mixture was left standing atroom temperature under vigorous stirring; after 15 min the pH wasadjusted at 7 with HCl 1M and the product purified from free drug andthe leaving group hydroxysuccinimid by gel filtration chromatography ona BIO GEL P 60 100-200 mesh column. The M-modified drug was eluted as apeak with the typical UV absorption of doxorubicine (OD 230 and 480 nm)and the expected iodine reaction for MPEG. The M-PEG5000-Gly-doxorubicin fractions were collected, concentrated byultrafiltration and lyophilized. The product was further purified bychromatography on a BIO GEL A 0.5 m column. The overall yield, based onthe starting drug, was 50%.

We claim:
 1. Biologically active drug polymer derivatives having the formula

    RO--(CH.sub.2 --CH.sub.2 O).sub.n --(CO)--NH--X--(CO)--NH--Z (I)

wherein R represents a lower alkyl group, n is an integer between 25 and 250, X when combined with adjacent NH and CO groups represents an amino acid or a dipeptide or tripeptide residue, and Z when combined with the adjacent NH group represents a biologically active peptide or protein or NH or NH₂ containing drug residue.
 2. Drug polymer derivatives according to claim 1 whereinR represents a methyl group, n is an integer between 40 and 115, X when combined with adjacent NH and CO groups represents an amino acid residue selected from the group consisting of glycine, phenylanaline, tryptophan and norleucine, or a dipeptide or tripeptide residue selected from Gly-Gly, Arg-Arg, Phe-Arg, Gly-Gly-Arg, Gly-Gly-Phe, and Gly-Leu-Gly-Leu, and Z when combined with the adjacent NH group represents the residue of a biologically active peptide, protein or drug selected from the group consisting of superoxidedismutase, ribonuclease, arginase, asparaginase, urokinase, ampicilline, doxorubicine, N-desmethyl-tamixofen, LHRH and synthetic analogues of same, somatostatin and synthetic analogues of same, calcitonin, interleukin-2, tumor necrosis factor, insulin, IGF-1, natural or recombinant interferon, adenin-arabinoside (ara-A), cytosin-arabinoside (ara-C) and acyclovir.
 3. Method for preparing biologically active drug polymer derivatives having the formula (I) as defined in claim 1 which comprises:a) reacting a mono-alkoxy-polyethylene glycol derivative of formula

    RO--(CH.sub.2 --CH.sub.2 O).sub.n H                        (II)

wherein R and n have the definition as indicated in claim 1 with 2,4,5-trichlorphenylchloroformate or 4-nitrophenylchloroformate to obtain the corresponding carbonate; b) reacting the carbonate thus obtained with an amino acid or a di- or tripeptide of formula

    H.sub.2 N--X--(CO)OH                                       (III)

wherein X is defined as indicated in claim 1 to obtain a compound of formula

    RO--(CH.sub.2 --CH.sub.2 O).sub.n --(CO)--NH--X--(CO)OH    (IV)

such that the carbonate is bonded directly to the amino group (--NH₂) of the amino acid or peptide; c) converting the compound of formula (IV) thus obtained into the corresponding succinimidyl ester; and d) finally, reacting the said succinimidyl ester with a biologically active peptide or protein or NH or NH₂ -containing drug of formula

    R--NH--Z or H2N--Z                                         (V)

wherein R and Z are defined as indicated in claim 1 such that the peptide, protein or drug is bonded to the activated carboxyl function of the ester.
 4. Pharmaceutical composition which comprises as an active ingredient at least one biologically active drug polymer derivative of formula (I) as defined in claim 1; and a pharmaceutically acceptable carrier.
 5. Pharmaceutical composition according to claim 4 which comprises as active ingredient a biologically active drug polymer derivative of formula (I) whereinR represents a methyl group, n represents an integer between 40 and 115, X when combined with adjacent NH and CO groups represents an amino acid residue selected from the group consisting of glycine, phenylanaline, tryptophan and norleucine, a dipeptide or tripeptide residue selected from Gly-Gly, Arg-Arg, Phe-Arg, Gly-Gly-Arg Gly-Gly-Phe and Gly-Leu-Gly-Leu, and Z when combined with the adjacent NH group represents a biologically active peptide, protein or drug residue selected from the group consisting of superoxidedismutase, ribonuclease, arginase, asparaginase, urokinase, ampicilline, doxorubicine, N-desmethyl-tamoxifen, LHRH and synthetic analogues of same, somatostatin and synthetic analogues of same, calcitonin, interleukin-2, tumor necrosis factor, insulin, IGF-1 natural or recombinant interferon, adenin-arabinoside (ara-A), cytosin-arabinoside (ara-C) and acyclovir.
 6. Pharmaceutical composition according to claim 5 which comprises as active ingredient a biologically active drug polymer derivative selected from the group consisting ofM-PEG 5000-Gly-superoxidedismutase, M-PEG 5000-Trp-superoxidedismutase, M-PEG 5000-nor-Leu-superoxidedismutase M-PEG 1900-Gly-superoxidedismutase, M-PEG 5000-Gly-arginase, M-PEG 5000-Gly-ribonuclease, M-PEG 5000-Gly-urokinase, M-PEG 5000-Gly-ampicillin, and M-PEG 5000-Gly-doxorubicin wherein M-PEG represents monomethoxy-polyethylene. 